Differential expression of genes is fundamental to biological processes. It is accomplished by the combinatorial and synergistic (or antagonistic) action of a relatively small number of transcription factors. To elucidate transcriptional regulatory mechanisms in the retina, it is essential to identify specific activators (and repressors) and delineate how these regulators integrate the signaling pathways with basal transcription machinery to generate a transcriptional response. We have previously identified Nrl, a transcription factor of the basic motif-leucine zipper (bZIP) family, by subtraction cloning. Nrl regulates rhodopsin promoter activity synergistically with Crx, a photoreceptor-specific transcription factor. Mutations in the human CRX or NRL gene result in photoreceptor degeneration, suggesting a major role for these two transcription factors in modulating photoreceptor gene expression in vivo. Our studies also reveal that Nrl interacts with Crx, TATA-binding protein, and other as yet uncharacterized proteins in the retina. Because of its unique expression pattern, possible regulation by FGF-2, and involvement in rhodopsin regulation, Nrl appears to be a key mediator of transcriptional response in the developing and mature retina. We hypothesize that interaction of Nrl with other regulatory proteins in the context of DNA-binding sequences is responsible for spatial and temporal control of gene expression in the retina. The goals of this proposal are to identify Nrl- interacting proteins (NIPs) in the retina using genetic (yeast two-hybrid) and biochemical (immuno- and DNA-affinity chromatography) methods, with a focus on delineating rhodopsin regulation (Aims 1-3). To assess the role of Nrl in developing retina, we propose to isolate NIPs from fetal retinal libraries by a yeast two-hybrid approach (Aim 2) and directly examine Nrl function in mice using a gene-knockout strategy (Aim 4). Elucidation of transcriptional regulatory pathways should reveal significant new insights into retinal development and disease. Since mutations in retinal transcription factors and their target genes result in retinopathies, it might be possible to experimentally manipulate the function of specific transcription factor(s) to up- or down-regulate a particular target gene and correct a disease phenotype.